The heart has evolved an adaptive response to stress, termed late preconditioning (PC), that confers powerful and sustained protection against ischemiaJreperfusion injury. Previous work by us and others has shown that late PC is mediated by the upregulation of a cluster of stress-responsive cardioprotective proteins, namely, iNOS, HO-1, and ecSOD. Our preliminary data indicate that these proteins are organized hierarchically and are inextricably linked, forming a functionally inter-related module. Building upon this previous work, we will determine the feasibility of transferring these genes to the heart to achieve a permanent preconditioned-like state. We will also determine the mechanism by which the respective proteins interact. All studies will be performed in a well-established murine model of myocardial infarction using recombinant adeno-associated virus (rAAV) vectors. In Aim 1, we will determine whether rAAV-mediated transfer of the genes that form the iNOS-HO-1-ecSOD module produces chronic cardioprotection and whether this is due to prevention of apoptosis, necrosis, or both. Mice will undergo iNOS, HO-1, or ecSOD gene transfer and then will be subjected to myocardial infarction 1, 6, or 12 months later to determine the presence of a protective phenotype. In Aim 2, we will decipher the interplay between iNOS and HO-I. Using both a loss-of-function (HO-1-/-) and a gain-of-function (HO-1 TG) approach, we will determine whether the infarct-sparing effects of iNOS gene therapy depend on the presence of HO-1 and whether HO-1 modulate iNOS and thus limits oxidative and nitrative stress after iNOS gene therapy. The specific role of CO (one of the by-products of HO-1 ) will be elucidated by using novel CO-releasing agents. In Aim 3, we will use ecSOD-/- mice to test the hypothesis that ecSOD is obligatorily required for iNOS-dependent, HO-1-dependent, and CO-dependent protection. We will also test the novel idea that HO-1 augments cardiac NO levels by upregulating ecSOD. Finally, in Aim 4 we will use Nrf2 -/- mice to test the hypothesis that Nrf2, a pleiotropic stress-responsive transcription factor, mediates iNOS-dependent upregulation of HO-1 and iNOS- and HO-l-dependent upregulation of ecSOD. These studies will test the hypothesis that Nrf2 is a critical "switch" that links iNOS, HO-1, and ecSOD. Throughout this Project, evidence for or against a functional role of specific proteins will be obtained by using genetic manipulations in intact animals (gene transfer, transgenic mice, or KO mice). This molecular physiologic strategy will make it possible to arrive at conclusions that are definitive, and at the same time, physiologically relevant. These studies will be the first to test the novel idea that gene therapy can be used to achieve chronic prophylactic cardioprotection. The results will provide a framework for translating cardioprotective gene therapy to the clinical arena and will have broad pathophysiological implications for numerous biologic processes in which NO and CO are known to serve an important regulatory function.